Project Summary Traumatic brain injury (TBI) causes detrimental behavioral dysfunctions and brain neurodegeneration. The problem in the brain injury field is that there are no effective therapeutic agents for TBI or TBI-related conditions. Advancements in new drug targets are necessary for new opportunities to discover effective therapeutics based on the target mechanism. We have identified cathepsin B as an effective drug target for TBI based on data showing that knockout of the cathepsin B gene results in substantial improvements in TBI- induced motor dysfunction and neuropathology in the CCI (controlled cortical impact) mouse model of TBI. Further, we have shown that the potent E64c inhibitor of cathepsin B ameliorates TBI-induced motor deficits and neuropathology. The inhibitor was administered orally as the prodrug E64d, which is converted to the active E64c drug inhibitor in vivo in the animal by esterases. The cathepsin B knockout mice are healthy, which indicates that inhibition of cathepsin B will be safe. Indeed, prior studies of E64d administered to pediatric patients was found to be safe, without toxicity. Based on this compelling data for cathepsin B as a novel drug target for TBI, and inhibition of cathepsin B for improving TBI deficits, the goal of this Phase I project will be to define the in vivo pharmaceutical properties of the E64c, after oral administration of the E64d prodrug, with respect to (a) pharmacokinetics (PK) and metabolite analyses in plasma and brain, (b) target engagement (TE) of E64c to cathepsin B in brain, (c) pharmacodynamics (PD) of cathepsin B mediated activation of the IL-1? pro-inflammatory cytokine, and (d) efficacy to improve motor dysfunction and neuropathology. While the efficacy of E64d to improve TBI deficits has been demonstrated, the pharmaceutical properties of E64d under the efficacious doses have not yet been determined. These research plans will assess the hypothesis that E64c possesses brain PK//TE/PD properties demonstrating efficacy to improve TBI-induced motor dysfunction. Aim 1 will determine the E64c properties of pharmacokinetics (PK), target engagement (TE), pharmacodynamics (PD), and general health after oral administration of E64d to the CCI TBI mouse. This aim includes novel TE mass spectrometry technology to quantitate the percent of cathepsin B bound to inhibitor drug. Aim 2 will evaluate efficacy of E64d (administered orally) for improving motor dysfunction and neuropathology in the CCI mouse model of TBI. Administration of the inhibitor several hours after CCI TBI injury will bet tested to provide a translatable time-frame for future clinical drug administration several hours after injury in medical emergency situations. Aims 1 and 2 will indicate the relationship of behavioral improvement with brain PK, TE, and PD properties of E64c, with dosing regimens of E64d. Findings will support the next Phase II plan to gain detailed understanding of the inhibitor drug properties to provide the necessary pre-clinical data for planning IND and clinical studies.